Optical detection and analysis of small molecules, bio-molecules, and cells can be broadly divided into two categories: absorption spectroscopy and emission spectroscopy. Absorption spectroscopy involves illuminating a sample with radiation over a range of wavelengths, such as over the visible and infrared portions of the electromagnetic spectrum, and measuring the intensity of the radiation transmitted through the sample. Subtracting the measured intensity from the intensity of the radiation incident on the sample gives the absorption spectrum, or amount of radiation absorbed by the sample as a function of frequency. In emission spectroscopy, incident radiation at a single wavelength causes the sample to fluoresce at one or more frequencies; the resulting plot of fluorescence intensity versus frequency is the sample's emission spectrum. Because each chemical compound has unique absorption and emission spectra, absorption and emission spectroscopy can be used to identify unknown samples.
Spectroscopic techniques can also be used to count, identify, and sort particles for purifying fluids and diagnosing disease. In flow cytometry, for example, particles are suspended in a fluid and passed through a beam of light. A detector senses the absorption or emission spectrum of the particles as they flow through the beam of light. High-throughput flow cytometry exploits a novel many-samples/one-file approach to dramatically speed data acquisition, to limit aspirated sample volume to as little as 2 μl/well, and to produce multi-sample data sets that facilitate automated analysis of samples in groups as well as individually. High-content multiparametric analysis capabilities have been exploited for assay multiplexing, allowing the assessment of biologic selectivity and specificity to be an integral component of primary screens. These and other advances in the last decade have contributed to the application of flow cytometry as a uniquely powerful tool for probing biologic and chemical diversity and complex systems biology.
Spectroscopic flow cytometry can be combined with polarimetric techniques to identify anisotropic and chiral particles, including different enantiomers of the same compound. As understood by those of skill in the art, an anisotropic particle has different dimensions along different axes—for example, an ellipsoid is anisotropic, whereas a sphere is isotropic. A chiral particle is a particle that lacks an internal plane of symmetry, i.e., a chiral molecule has a non-superimposable mirror image. Chiral particles are anisotropic, but anisotropic particles are not necessarily chiral.
The ability to distinguish anisotropic and chiral particles from isotropic particles is especially useful when sorting an active isomer from a detrimental or inactive isomer of a drug. In some cases, a Pockels cell or other device switches the polarization of the beam of light used to irradiate the particles in the flow cytometer between linearly polarized and circularly polarized states. Differences between the emission or absorption spectra for the different polarization states may indicate the presence of different isomers of the same compound in a given sample. Similarly, changes in the amount of scattered light and the direction in which the light is scattered as a function of polarization state may indicate the presence of particles with different shapes (e.g., isotropic versus anisotropic).
Recognizing the chirality of chemical and biological compounds is especially important for identifying different isomers of a single molecule and of chiral macromolecules, including proteins, DNA, and various metabolites. Chirality in nanotechnologies is also important in applications such as functional self assembly, enantioselective catalysis, separation, biosensing, and optical devices. There are many biological systems at microscopic and macroscopic levels which are enriched by chiral objects such as proteins, nucleic acids, carbohydrates, amino acids, and nucleotides.